Semester

Spring

Date of Graduation

2011

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Biology

Committee Chair

Clifton P Bishop

Abstract

The techniques currently utilized for collecting biological evidence at the scenes of violent crimes such as murder, rape, and kidnapping are employed simply to preserve evidence for future DNA analysis. Although DNA is extremely useful in forensic casework, biological samples possess yet another valuable molecule, RNA. The use of RNA in forensic analysis of samples is currently on the rise. In the past, RNA was thought to degrade much too quickly to be of any benefit to the forensic community. The work presented in this dissertation seeks to demonstrate otherwise. While true that RNA is not as stable as DNA, its rate of degradation in ex vivo specimens has been found to be a function of the size and species of the molecule, as well as predictable in certain biological fluids and tissues. The first objective of this research was to determine if the size-dependent, predictable decay of RNAs could be utilized as an indicator of postmortem interval (PMI), or time since death. A method to efficiently isolate and correctly quantify RNA from tooth pulp was established. By quantifying the remaining molecules of a large versus a small section of beta-actin mRNA from aging tooth pulp from deceased pigs, and the postmortem alterations in pulp color, a relationship between these factors and PMI was established. Previous studies had indicated that rate of RNA decay may be a temperature-dependent process. It was determined that the degradation rate of RNA could be better described by the accumulation of temperature to which pigs were exposed, rather than time in days. This analysis led to a simple mathematical equation into which measurable variables can be placed to find the number of accumulated degree days (ADD) that have passed since death with 95% confidence. The resulting method can provide reliable estimates of PMI that surpass the amount of time following death in which current estimators, primarily forensic entomology, can reliably produce an estimate of time since death. We have also developed a novel technique to determine the body fluid type of biological stains found at the scene of violent crimes. This technique makes use of fluorescently labeled probes known as molecular beacons (MBs) to identify tissue-specific RNAs in a confirmatory assay. Importantly, it has the potential to be made portable to a crime scene and does not involve the use of PCR. It was successfully demonstrated that the MBs designed for experimentation release a signal only in the presence of RNA extracted from one of three tissues: blood, saliva, or semen. By determining if a potential piece of evidence should be collected or left behind at the scene as not pertinent to the crime at hand, this technique has the potential to reduce DNA backlog among crime laboratories.

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